316 Fungal Enzymes

13
Pectinases Produced by
Microorganisms: Properties
and Applications
Maria de Lourdes Teixeira de Moraes Polizeli,1,*
André Ricardo de Lima Damásio,2,a Alexandre Maller,2,b
Hamilton Cabral,3 Aline Moraes Polizeli 1 and Mahendra Rai4

Introduction
At present, microbial enzymes are being extensively used in different areas,
due to their high efficiency and specificity (Damásio et al. 2010). Microbial
enzymes have found applications in areas of genetics, protein engineering
and in the development of new industrial processes, including processes

1
Biology Department, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, São Paulo
University, Brazil.
E-mail: polizeli@ffclrp.usp.br; alinepolizeli@gmail.com
2
Biochemical and Immunology Department of School of Medicine of Ribeirão Preto, São
Paulo University, Brazil.
a
E-mail: andre.damasio@gmail.com
b
E-mail: alemaller@yahoo.com.br
3
Science Pharmaceutical Department of School of Pharmaceutical Science of Ribeirão Preto,
São Paulo University, Brazil.
E-mail: hamilton@fcfrp.usp.br
4
Department of Biotechnology, SGB Amravati University, Maharashtra, India.
E-mail: mkrai123@redifiimail.com
*Corresponding author
 Pectinases Produced by Microorganisms: Properties and Applications 317

related to the environmental pollution control. Pollution is no longer seen

by society as an acceptable problem. Among enzymes that show intensive
applications in industrial and environmental sectors, it is those from the
pectinolytic system that act on pectin or structurally related compounds.
The term “pectin” comes from Greek, and means clotted. It is a natural
hydrophilic colloid found in the middle lamella and primary wall of higher
plants, where it does not exceed 1 percent of their fresh mass (Rombouts
and Pilnik 1980, Hugouvieux-Cotte-Pattat et al. 1994). This term designates
a mixture of several compounds, of which the main is pectinic acid. Its
active form is located in the cell wall and may be interconnected to other
structural polysaccharides and proteins, forming insoluble protopectin
(Kashyap et al. 2001). The pectic chain is involved in functions related to
physiology, growth and plant development. In green fruits, pectin is found
in its insoluble form (protopectin), associated to cellulose microfibrils,
giving rigidity to the cell walls. During fruit ripening, enzymes hydrolyze
the pectin chain or its lateral chains, making them to become more soluble
(Willats et al. 2001).
Pectic substances are degraded by pectinases or pectinolytic enzymes,
which are produced by a large number of bacteria, yeasts and fungi,
insects, nematodes and plants (Whitaker 1991). These enzymes degrade
pectin of the middle lamella resulting in tissue disintegration in order to
obtain a source of carbon or a modification during fruit maturation. Pectin
degradation of the middle lamella results in the degradation of tissue by
cell separation, a process denominated maceration (Bailey and Pessa 1990).
Due to the general occurrence of pectic polysaccharides in many vegetables,
the enzyme systems capable of degrading them have various mechanisms
of action (Rombouts and Pilnik 1980, Kashyap et al. 2001).
Innumerable applications of the enzymes that act on the pectinolytic
system stimulate the search for new microorganisms and carbon sources that
can act as genetic inducers of the pectinase synthesis in cells. Application
in extraction and clarification of fruit juices and vegetables, extraction of
olive oil, wine production, animal feed, textile industry, cellulose and paper
industries, functional foods and pharmaceuticals, fermentation of tea and
coffee, treatment of wastewater pectin and treatment of the banana fiber
will be discussed.
This chapter describes recent advances related to the complex structures
of pectins, classification of pectinases and their mechanisms of action,
production by microorganisms and applications in a number of industrial
and environmental control processes.
318 Fungal Enzymes

Pectin Structure
The pectin molecule is included in the family of complex polysaccharides,
having a molecular mass between 30 and 300 kDa. It consists of a main
chain of 1,4-α-D-galacturonic acid (GalA), partially esterified with methanol
or acetyl groups (Villa 1999); its methanolic groups are partly substituted
by sodium, potassium or ammonium ions (Fig. 13.1). Some sugars like
L-rhamnose, D-galactose, D-xylose and L-arabinose may take part in
interrupting the galacturonic chain (Fogarty and Ward 1974, Fogarty and
Kelly 1983). The esterification level, the proportion of neutral saccharides
and the degree of polymerization are the main heterogeneity elements
found in pectic compounds of different origins.
Three great groups of pectic polysaccharides (homogalacturonan,
rhamnogalacturonan-I and rhamnogalacturonan-II) have been isolated
from plant primary cell walls and structurally characterized (Ridley 2001,
Willats et al. 2006) (Fig. 13.1).
Homogalacturonan (HG) consists of a linear chain of GalA, linked by α-1-4
linkages meaning that D-GalA in the HG polymer can be esterified and/
or acetylated. The proportions of methyl esterification and acetylation
have a profound impact on functional properties. Pectins are traditionally
categorized as much or little esterified, when showing methyl-esterification
levels higher or lower than 50 percent, respectively (Vincken et al. 2003). A

Figure 13.1 Representation of the structures of pectin. Dha: Deoxylyxoheptulopyranosylaric

acid, KDo: Ketodeoxymannooctulopyranosylonic acid.
Color image of this figure appears in the color plate section at the end of the book.
 Pectinases Produced by Microorganisms: Properties and Applications 319

pectin gel is formed when the portions of HG are interconnected to form

three-dimensional crystalline net retaining water and solutes.
Rhamnogalacturonan I (RGI) consists of a repeating chain of alternating
GalA and rhamnose, which may have lateral chains of neutral residues
like galactose, arabinose and xylose. Some stretches consist of alternating
galacturonic acid and rhamnose—“hairy regions”, others of lower density
of rhamnose—“smooth regions”.
Rhamnogalacturonan II (RGII) has a structural support of HG and unlike
RGI, complex lateral chains are connected with GalA residues. Until
recently, it was accepted that the domains of rhamnogalacturonan and
homogalacturonan constitute the structural basis of the pectin polymers.
However, an alternative structure has recently been proposed in which
a long chain of RGI forms the structure of the molecules (Fig. 13.1). The
composition of the RGI sugars may be highly heterogeneous; on the other
hand, RGII has a highly preserved structure (Vincken et al. 2003).
Several factors determine the jellifying properties of pectins, including
temperature, type of pectin, pH, and presence of sugars, calcium and other
solutes. In highly esterified pectins, binding zones are formed by crosslinks
of HGs through hydrogen bonds and interactions between methoxyl groups,
both resulting from the high concentration of sugar and an acid pH. In pectins
with a lower number of esterifications, junctions are formed by crosslinks
between calcium ions and free carboxyl groups (Willats et al. 2006).
Based on the type of modifications of the bounds of the main chain,
pectic substances may be classified into protopectin, pectic acid, pectinic
acid and pectins. Information on each of these terms is provided below.
- Protopectin is a term occasionally used to describe water-insoluble
pectic substances, connected to cellulose fibers, found in plant tissues
and utilized to produce soluble pectic substances. Pectic acids are
galacturonan containing small quantities of methoxy groups.
- Pectic acid salts are called pectates.
- Pectinic acids are galacturonans with various amounts of methoxy groups.
They possess the property of forming a gel with sugars and acids.

Pectinase Classification
Pectinase classification is based on their action on pectin molecule
(Fig. 13.2). As recommended by the Committee on Pectin Nomenclature
(1944), pectinolytic enzymes are generically called pectinases and
basically may be divided into three classes according to the following
criteria (Kashyap et al. 2001, Sakai et al. 1993, Hoondal et al. 2002, Uenojo
et al. 2007):
320 Fungal Enzymes

Figure 13.2 Pectinases and action sites on pectin structures. (A) PG: polygalacturonase, PMG:
polymethylgalacturonase; (B) PGL: polygalacturonate lyase, PMGL: polymethylgalacturonate
lyase; (C) PMGE: polymethylgalacturonate esterase.

(1) Pectinesterase (PE), (EC 3.1.1.11; PE): catalyzes the de-esterification

of pectin methoxyl groups forming pectic acid and methanol. It
acts preferably on the methoxyl ester group of a non-esterified
galacturonic unit (Gummadi and Kumar 2005). It can also be classified
as (PME) pectin methyl esterase, pectin demethoxylase and pectin
methoxylase.
(2) Depolymerases: can also be characterized according to the type
of cleavage (hydrolysis or trans-elimination) and also to the mode
of action on the substrate molecule, that is, by random cleavage of
glycosidic bindings in the interior of the molecule (endo-activity) or
by cleavage of terminal bindings (exo-activity).
 Pectinases Produced by Microorganisms: Properties and Applications 321

Hydrolases act by splitting glycosidic bonds between galacturonic units by

the entrance of a molecule of water and can be classified as:
- Endopolygalacturonase (pectin depolymerase, EC 3.2.1.15, endo-
PG): randomly cleaves glycosidic α-1,4 bonds of pectate and other
galacturonans;
- Exopolymethylgalacturonase (poly (galacturonate) hidrolase, EC
3.2.1.67; exo-PG): catalyses sequentially the hydrolysis of the glycosidic
α-1,4 bonds of pectate and other galacturonans and acts at the chain
terminal.
The lyases act through trans-elimination forming a double bond between
C-4 and C-5 of the monomer released from the substrate. Lyases can be
classified in:
- Endo-pectin lyase (or pectin methyltranseliminase; pectin
transeliminase, pectolyase; poly methylgalacturonic transeliminase, EC
4.2.2.10): cleaves randomly pectate and other galacturonas producing
unsaturated methyl ester oligogalacturonates;
- Exopolymethylgalacturonate lyase (poly (methoxygalacturonide)
exolyase; exo-PMGL): causes sequential cleavage of the pectin molecule
through a transelimination process;
- α-1,4-D-Endopolygalacturonic acid lyase or endopectate lyase—EC
4.2.2.2; endo-PGL): randomly cleaves pectate and little methoxylated
pectin, producing dimers and trimers and a series of unsaturated
oligogalacturonates;
- Exopolygalacturonate lyase (or exopectate lyase—EC 4.2.2.9): cleaves
sequential α-1,4 bonds of pectic acid, releasing unsaturated products
from the reducing end.
(3) Protopectinase: solubilizes protopectin resulting in highly polymerized
pectin.
Complete degradation of pectin involves the action of RG
rhamnohydrolases (Fig. 13.3), that promote depolymerization of the
rhamnogalacturonan chain, releasing rhamnose from the non-reducing
ends, RG galacturonohydrolases that release monogalacturonic acid from
the non-reducing chain ends (Mutter et al. 1998), RG acetylesterases that
hydrolyze acetylgroups of rhamnogalacturonan (Searle-Van Leeuwen et al.
1992) and xylogalacturonases that cleave the bond between galacturonate
residues and xylose releasing xylose-galacturonic acid dimers (van der
Vlugt-Bergmans et al. 2000).
Polygalacturonases can also be classified into the family 28 of glycoside
hydrolases (Table 13.1). In this family, there are also endopolygalacturonases,
exopolygalacturonases, RG rhamnopolygalacturonases and endoxylogalacturane
322 Fungal Enzymes

Figure 13.3 Enzymes and action sites on rhamnogalacturonan II structure.

Color image of this figure appears in the color plate section at the end of the book.

hydrolases. These enzymes have a catalytic site of an aspartate residue

functioning as a nucleophile and another one that acts as a proton donor.
Family 1 of polysaccharide-lyases harbors endopectate lyases, exopectate
lyases and pectin lyases. Families 1 and 28 present helical beta parallel
structural motives.

Pectinases from Microorganisms

Microbial pectinases have occupied a prominent place in industrial
enzymology. Bacteria, yeasts and fungi can produce pectinases. Among
reported microorganisms are Fusarium solani, Aureobasidium pullulans,
Selenomonas tuminantium, Aspergillus niger, Penicillium griseoroseum, Candida
 Pectinases Produced by Microorganisms: Properties and Applications 323

Table 13.1 Classification and structure of pectinases families.

Family Known activities 3D Structure Status EC number

1
GH -28 endopolygalacturonase Fold: (β)-helix 3.2.1.15
GH-28 exopolygalacturonase Fold: (β)-helix 3.2.1.67
GH-28 exo-polygalacturonosidase Fold: (β)-helix 3.2.1.82
GH-28 rhamnogalacturonase Fold: (β)-helix 3.2.1.-
PL2-1 endopectate lyase Fold: parallel β-helix 4.2.2.2
PL-1 exopectate lyase Fold: parallel β-helix 4.2.2.9
PL-1 endopectin lyase Fold: parallel β-helix 4.2.2.10
CE3-8 pectin methylesterase Fold: (β)-helix 3.1.1.11
CE-12 pectin acetylesterase non-classified 3.1.1.-
CE-12 hamnogalacturonan acetylesterase non-classified 3.1.1.-
CE-13 pectin acetylesterase non-classified 3.1.1.-
1
GH: Glicosyl hydrolases
2
PL: Polysaccharide lyase
3
CE: Carbohydrate esterase
Source: http://www.cazy.org/

macedoniensis, Fusarium oxysporum, Saccharomyces fragilis, Bacillus pumilus,

Bacteroides thetaiomicron and Rhizoctonia fragarie (Schuster et al. 2002,
Sathyanarayana and Panda 2003).
The genes that encode the pectinases are cloned and expressed in
various cell types. Some of the genes of endopolygalacturonases are cloned
and expressed in Saccharomyces cerevisiae (Jia and Wheals 2000), in Pichia
pastoris (Blanco et al. 2002), Escherichia coli (Wubben et al. 1999) and pectate
lyase in E. coli (Zhao et al. 2007a,b).
Pectinases present different specificities for substrates and properties
(Table 13.2), but basically can be separated into homogalacturonan and
rhamnogalacturonan groups. Pectinases produced from A. niger are the
most extensively used in industry. This fungus produces many enzymes
which are active on homogalacturonan, including pectin methyl- and
acetyl-esterase, endopolygalacturonase, exopolygalacturonase, pectate lyase
and pectin lyase. It possesses a complete family of PG codifying genes and
produces isoenzymes with considerable differences in relation to substrate
specificity, cleavage patterns and optimal activity of pH (Lang and
Dörnenburg 2000).
The biotechnological potential of fungal pectinolytic enzymes has
aroused the attention of several researchers. Several agro-industrial residues
like wheat bran, sugar-cane bagasse, coffee pulp, corncobs, lemon and apple
peels are being explored as inducers of microbial pectinases (Sathyanarayana
and Panda 2003, Patil and Dayanand 2006). A polygalacturonase was
produced from Aspergillus niveus cultured on liquid or solid media
supplemented with agro-industrial wastes (Maller et al. 2011). Submerged
Table 13.2 Biochemical properties of some pectinases from microorganisms.
324

Producer Enzyme pH# Temperature# (°C) Km* (mg.ml–1) Vmax Reference

Aspergillus niveus Exo-PG 4.0 55 - - Maller et al. 2011
Aspergillus niveus PL 8.5 55 - - Maller et al. 2012
Aspergillus niger NRRL3 PGI 5.0 40 - - Fahmy et al. 2008
Bacillus pumilus BK2 PGL 8.5 70 0.24 0.72 (c) Klug-Santner et al. 2006
Bacillus sp. TS47 PG 8.0 70 - - Takao et al. 2000
Fungal Enzymes

Fusarium moniliforme PGI 4.8 45 0.110 111.11 (a) Niture and Pant 2004
Fusarium moniliforme PGII 5.3 40 0.166 13.33 (a) Niture and Pant 2004
Kluyveromyces wickerhamii crude pectinase 5.0 50 - - Moyo et al. 2003
Mrakia frigida PGL 8.5–9.0 30 - - Margesin et al. 2005
Mucor flavus PGL 3.5–5.5 45 - - Gadre et al. 2003
Neurospora crassa PG 6.0 45 5.0 357 (b) Polizeli et al. 1991
Neurospora crassa exo-1 mutant PGI 5.5 40 1.161 177.94 (a) Crotti et al. 1998a
PGII 5.5 45 0.023 2.08 (a)
PGL 9.0 50 0.50 273.20 (a)
PL 10.0 50 0.076 363.40 (a)
Paecilomyces variotii Exo PG 4.0 65 1.84 432 Damásio et al. 2010
Penicillium frequentans PGI 3.9 50 0.68 596.8 (a) Chellegatti et al. 2002
Penicillium oxalicum PL 8.0 50 1.1 - Yadav and Shastri 2007
Rhizopus microsporus var. PG 3.5 65 - - Damásio et al. 2011
rizopodiformis
Streptomyces lydicus PG 6.0 50 1.63 677.8 (a) Jacob et al. 2008
Tetracoccospo-rium sp. PG 4.3 40 3.23 0.15 (b) Aminzadeh et al. 2006
Trichoderma harzianum PGII 5.0 40 3.4 1.28 (a) Mohamed et al. 2006
#
Optimum pH and Temperature
*This values of Km refer to use of sodium polypectate;
Symbols: (PG) polygalacturonase; (PL) pectin lyase; (PGL) pectate lyase; (a) µmol.min−1.mg−1;
(b) µmol.min–1; (c) g.l–1.min–1
 Pectinases Produced by Microorganisms: Properties and Applications 325

fermentation (SbmF) was tested using Czapek media supplemented with

28 different carbon sources. Among these; orange peel was the best PG
inducer. On the other hand, for solid state fermentation (SSF), lemon peel
was the best inducer. By comparing SbmF with SSF, both supplemented with
lemon peel, it was observed that PG levels were 4.4-fold higher under SSF.
Pectinase production can be induced by pectins of various sources, sodium
polypectate and polygalacturonic acid. These complex polysaccharides
can not enter the cells polysaccharides cannot enter the cells to affect gene
expression. Thus, the real inducers of pectinolytic enzymes are considered
compounds of low molecular mass or monogalacturonic acid itself.
There are reports on the role of the cell surface in the transduction of
regulatory signals that control the adaptation of fungal cells of the wild type
and mutants with mycelial phenotypes, and cell wall-less mutants (slime)
of Neurospora crassa to their nutritional medium (Polizeli et al. 1990). These
authors observed that the synthesis of cellulases, pectinases and xylanases
was induced by polysaccharide substrates. Carbon catabolic repression was
observed for all mutants with mycelial phenotypes. On the other hand,
for some slime mutants, all enzymes that degrade polysaccharides were
constitutively produced and were resistant to catabolic repression.
Polizeli et al. (1991) investigated pectinase production in the exo-1 mutant
of N. crassa, an hyperproducer strain that produces many exoenzymes and
also presents deficiencies in the cell wall composition (Gratzner and Sheehan
1969). This mutant secreted five to six times higher pectinase levels than
the wild type. This enzyme was characterized as a monomeric glycoprotein
with a high capacity to hydrolyze sodium pectate, characterizing it as an
endopolygalacturonase. The exo-1 mutant also induced pectinases with
galactose (Crotti et al. 1996, Crotti et al. 1998a, Crotti et al. 1998b). When
these authors compared the induction of pectinases produced by the exo-1
of N. crassa in the presence of monogalacturonic acid, galactose and pectins,
it was shown that polygalacturonase was induced preferably by galactose,
but galacturonic acid was the best inducer of lyase activity.
The inducing effect of galactose and galacturonic acid appeared to
differ:
(i) The mixture of galacturonic acid and galactose synergistically increased
the production of pectic enzymes, when compared to those obtained
by the inducers separately;
(ii) The inducing effect of galacturonic acid was partly inhibited by
glucose;
(iii) The inducer effect of galactose was increased in the presence of
glucose.
326 Fungal Enzymes

Taken together, these results suggest a more complex mechanism of

pectinolytic enzymes regulation by pectin-containing molecules.

Heterologous Expression of Fungal Pectinases

Filamentous fungi are widely used for the production of homologous and
heterologous proteins, but compared to homologous proteins, production
levels of heterologous proteins are usually lower. The production levels of
heterologous proteins have been drastically improved (Gouka et al. 1997).
Homologous and heterologous protein production by filamentous fungi is
often limited by the expression of proteases at high levels. By eliminating
specific protease activities, protein production in filamentous fungi can be
considerably improved (van den Homberg et al. 1997).
The production of pectinases has been improved by heterologous
expression, mainly in filamentous fungi. A rhamno galacturonan
acetylesterase (RGAE) was purified to homogeneity from the filamentous
fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was
determined by Kauppinen et al. (1995). Full-length cDNAs encoding the
enzyme were isolated from an A. aculeatus cDNA library using a polymerase
chain reaction-generated product as a probe. The 936-base pair rha1 cDNA
encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino
acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus
oryzae, a filamentous fungus that does not possess RGAE activity, and the
recombinant enzyme was purified and characterized.
From the onset of gene technology, yeasts have been among the most
commonly used host cells for the production of heterologous proteins. At
the beginning of this new development, the attention in molecular biology
and biotechnology focused on the use of the best characterized species,
Saccharomyces cerevisiae, leading to an increasing number of production
systems for recombinant compounds (Gellissen and Hollenberg 1997).
A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned
by genomic Southern hybridization with a probe of a DNA fragment
synthesized by PCR. This was done using primers constructed based on the
N-terminal amino acid sequence of a polygalacturonase, protopectinase-
AS, produced by the strain and consensus internal amino acid sequence of
fungal polygalacturonases. The cloned gene was inserted into an expression
plasmid for yeast, pMA91, and introduced into S. cerevisiae to be expressed
(Nagai et al. 2000). A pectinolytic industrial yeast strain of S. cerevisiae was
generated containing the S. cerevisiae endopolygalacturonase gene (PGU1)
constitutively expressed under the control of the 3-phosphoglycerate kinase
gene (PGK1) promoter. The new strain contains DNA derived exclusively
from yeast and expresses a high polygalacturonic acid hydrolyzing activity.
 Pectinases Produced by Microorganisms: Properties and Applications 327

Yeast transformation was carried out by an integrative process targeting

a dispensable upstream region of the acetolactate synthase locus (ILV2),
which determines sulfometuron methyl resistance (Fernández-González
et al. 2005).
Purified proteins are a starting point for biophysical and biochemical
characterization methods that can assist in the elucidation of function. The
challenge for the production of proteins at the scale and quality required
for experimental, therapeutic and commercial applications has led to
the development of a diverse set of methods for heterologous protein
production.
Bacterial expression systems are commonly used for protein production
as these systems provide an economical route for it and require minimal
technical expertise to establish a laboratory protein production system
(Zerbs et al. 2009).
Pectin lyases from A. oryzae and A. niger are usually used for the
production of traditional fermented foods, but these fungi produce less
pectinases under natural conditions. The cDNA coding mature Pell (without
signal peptide) was amplified from A. oryzae by RT-PCR. Pell cDNA was
cloned into pET-28a (+) expression vector, then was transformed into
E. coli Turner (DE3) plac I cells to express Pell with 6-His tag. For improving
the efficiency of Pell expression in E. coli, the conditions of expressing the
Pell in E. coli were optimized. E. coli Turner (DE3) plac I cells with pET-28a
(+)-pell was first cultivated at 37ºC, 220 r/min until OD600 reached about
0.8. Then, cultivation broth was added with 0.05–0.1 mmol/L IPTG and
continuously incubated at 15ºC, at 170 r/min for 60 hr for expressing of Pell.
The recombinant expressed Pell activity could reach 400 u/mL medium,
which is 4000-fold of Pell produced naturally by A. oryzae and superior than
known recombinant amount of pectin lyases expressed in different fungi
expression systems (Zhao et al. 2007a,b).

Polygalacturonase Action Mechanism

Through the alignment of primary sequences of endopolygalacturonases,
there were four regions conserved Asn-Thr-Asp, Gly-Asp-Asp, Gly and
Gly-His-Arg-Ile-Lys (Pickersgill et al. 1998). In addition to these, other
clusters were detected by Sony and Ponnuswamy (2007)—Ser-Ile-Gly-Ser.
These regions are considered as the functional conservation (Sony and
Ponnuswamy 2007).
The catalytic sites of endopolygalacturonases have basically two
functions: the binding of substrate and its subsequent catalysis. According to
these shares, some catalytic mechanisms were determined. The glycosylated
hydrolases present two types of mechanisms, retention or inversion of
configuration of the anomeric carbon (Armand et al. 2000).
328 Fungal Enzymes

To understand the role of the catalytic-amino acid residues, site-directed

mutation in the fungal polygalacturonase was performed and suggested
that three aspartic acid residues Asp-180, Asp-201 and Asp-202 (numbered
according to the A. niger Polygalacturonase II) are directly involved in the
catalysis (Fig. 13.4A). In addition to these residues, Arg-256, Lys-258 and
His-223 (Fig. 13.4B) are also involved in the catalysis: the Arg and Lys
residues bind the substrate and His is the maintenance of state ownership
of ionizing the catalytic aspartic acid (Sony and Ponnuswamy 2007).

Figure 13.4 Ribbon representation of the conserved amino acid residues of the catalytic site
of Polygalacturonase of Aspergillus niger: (A) representation of Asp-180, 201 and 202; (B)
representation of the catalytic site residues. (The figure was generated using the ViewerLite
(Accelrys Inc.) program, based on the authors van Santn, Y. et al. 1999 and Pagès, S. et al. 2000.
The sequence using was 1czf PDB.)
Color image of this figure appears in the color plate section at the end of the book.
 Pectinases Produced by Microorganisms: Properties and Applications 329

Polygalacturonases acting by invertion mechanism have a pair of

carboxilic acids in the catalytic site, where one of them acts as a proton
acid releasing oxygen to the glycosidic linkage and the other acts as a base
activated by a water molecule, thus making the nucleophilic attack on the
sugar anomeric carbon (Sony and Ponnuswamy 2007).
In the enzymes acting by an inverted mechanism, the glycosidic linkages
are hydrolyzed in water by a simple mechanism of direct displacement in
the anomeric center. The glycosidases showing the retention mechanism
involves the formation of an intermediary (glycosyl-enzyme), in which
the sugar is covalently linked to the protein via the carboxylic side chain
of aspartic or glutamic acid, and the covalent bond-glycosylated enzyme
(glycosyl-enzyme) is formed and hydrolyzed (Withers 1995).
For a better understanding of the Polygalacturonase II catalytic
mechanism of the fungus A. niger (Armand et al. 2000), we performed
site-directed mutations of six amino acid residues conserved between
Polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256 and Lys-
258). All mutated residues are apparently critical for the catalysis, except
for the His-223.
The direct involvement of His-223 in the active site of polygalacturonase
II, according to Armand et al. (2000) can be a residue to make the donation
of proton in the reaction catalyzed by polygalacturonase II.
When the mutation in His-223 was concluded, it was observed that
the catalysis was affected, but the frequency at which the cleavage of the
connection occurred was not changed dramatically when the Histidine was
replaced by Cysteine at position 223. This mutation did not cause disruption
in subsites –1 and +1, indicating that His-223 plays a role in the catalysis
by maintaining an optimal state of ionization of the carboxylate involved
in the catalysis by protons share. This fact is corroborated by the fact that
the mutated enzyme in the CYS-223, whose sulfhydryl group is capable of
sharing proton, had its activity remaining high (Armand et al. 2000).
The role of aspartic acid in the hydrolysis of glycosidic linkages is
confirmed, for example, in the inverted mechanism, where two groups are
required for a distance of carboxylic 10Å (± 2) (Armand et al. 2000).
The Asp-201 in general makes the protonation of the products when they
are hydrolyzed; there are three assumptions supporting this proposal:
I) When the mutation of this residue occurred, it was possible to see that
the enzyme became inactive;
II) The mutation of this residue showed a minor effect on BCFs (Bound
Cleavage Frequencies) of the enzyme on olygogalacturonates,
suggesting that Asp-201 is not directly interacting with the
substrate;
330 Fungal Enzymes

III) The His-223 residue is fundamental for the catalysis. The His-223 shares
protons with Asp-201, allowing this last residue to be in the ionization
state appropriate to protonate the product (Armand et al. 2000).
After the resolution of the structure from these three aspartic acid
residues of polygalacturonase from Erwinia carotovora, the amino acids
corresponding to Asp-180 and Asp-201 in PGII are directly involved in
catalysis, according to Pickersgill et al. (1998).

Applications of Microbial Pectinases

Microbial enzymes are usually applied in many industrial and environmental
sectors. The world enzyme market has a commercial annual turnover of
US$2 billion dollars and keeps growing every year at a rate of 8–10 percent
(data supplied on-line by the Brazilian Ministry of Science and Technology).
The food industry has benefited mostly from the use of enzymes, using 15
percent of industrial processes. Furthermore, this sector has been showing
an increasing expansion of 9.2 percent in relation to 2004 (Association of
Biological Agriculturists of the State of Rio de Janeiro, Brazil). From this
total, the industry of natural juices, the largest source of application of
pectinases, had a production of 11 million liters between January and March
of 2007, a 12.6 percent growth relative to the same period of 2006 (Brazilian
Association of Soft Drinks and Non-Alcoholic Drinks).
Pectinases began to be used in the food industry in 1930 (Kertesz 1930).
Following technological advances and the need for new sources of fungal
pectinases, they became one of the most important industrial enzymes,
acquiring high application in textile processes, degumming of plant fibers,
treatment of aqueous residues, tea fermentation, food, beer, wine, animal
feed, pulp, paper industries and agriculture (Bajpai 1999). A more detailed
description of pectinase participation in various industrial sectors is given
below.

Extraction and clarification of fruit juices and vegetables

Most preparations of microbial pectic enzymes are used in industrial
processes utilizing fruits; even so, these represent only about a quarter of
the production of enzymes used in food worldwide. Around 1930, when
the food industries started juice production, yields were very low and many
difficulties arose for their filtration and clarification. Thereafter, research on
pectinases, cellulases and hemicellulases of the fungi A. niger and Trichoderma
sp. together with the increasing knowledge on fruit composition, helped to
overcome these difficulties (Grassin and Fauquembergue 1996).
 Pectinases Produced by Microorganisms: Properties and Applications 331

Usually pectinases (pectin lyase, methylesterase, endo and

exopolygalacturonases, pectin acetylesterase, rhamnogalacturonase,
endo- and exoarabinases), combined with cellulases and hemicellulases
—collectively called maceration enzymes—are used for the extraction and
clarification of fruits and vegetables (Grassin and Fauquembergue 1996,
Galante and Conti 1998, Gummadi and Panda 2003).
During the production of apple and pear juice, all the fruits are ground,
forming a mass, which after a mechanical process, yields a limpid juice and a
solid phase called pulp (Galante et al. 1998). The use of maceration enzymes
increases both yields as well as initial processes without high investments.
Maceration enzymes are generally used in two steps:
(1) following grinding, to macerate the fruit pulp until partial or full
liquefaction, that not only increases yields, decreases periods for
processing, but also increases the extraction of nutritive components
from the fruit;
(2) following juice extraction, pectinases are used for clarification, therefore
decreasing viscosity prior to concentration, increasing filtration rates
and the stability of the final product.
Commercially produced juices by these industries include: sparkling
ones (apple, pears and grapes), turbid juices (citric, plums, tomato, and
nectars) and unicellular products (resulting from plant tissue transformations
in a suspension of intact cells), whose objective is to preserve cell integrity
by selective hydrolysis of middle lamella polysaccharides (Kashyap
et al. 2001).
Unlike apple pectin that is highly methylated, orange juice naturally
contains a large amount of pectinesterases that cleave methoxyl groups from
pectin molecules. In the presence of Ca++ ions, insoluble calcium pectinate
is formed, resulting in undesirable particle precipitation. To prevent this,
two methods are available: the first is to denature the pectinesterases by
heating the juices; however, this procedure interferes with the product
flavor. An alternative is to freeze the juice concentrate, keeping the enzyme
in an active state.
Juices are in general commercialized in this form; however, concentrating
and freezing are very expensive for storage and transportation. Pectinases
can be used in two ways in the extraction of fruit juice: at the end of pulp
extraction (0.5 ± 2.0 g/100 Kg pulp at room temperature), to reduce viscosity
or right after finishing its manufacturing at the same concentration, at room
temperature, for 30 min (Rebeck 1990). This enables better extraction of
sugars and soluble solids, resulting in higher yields and lower viscosity.
The traditional method of clarification of lemon juice uses the natural
presence of pectinesterases. Compounds responsible for turbidity are
mainly obtained from fruit peels. Peel and pulp are ground into 3–5 mm
332 Fungal Enzymes

particles, mixed with water (1:1), heated to 95ºC to destroy methylesterases

and cooled to 50ºC. The mixture contains large amounts of pectin, other
polysaccharides, glycoproteins and essential oils; it is then treated with
pectinases or pectinases plus cellulases. After 1 hour, until the end of the
process; the liquid extract, if necessary, is again depectinized with the
enzyme. Centrifugation, pasteurization, concentration, bottling and sales,
follow.
Pectinases play an important role in tea and coffee fermentation.
The latter uses pectinolytic enzymes to remove the mucilaginous layer
present in the grains. Sometimes pectic enzymes are used to remove the
mucilaginous coat from the grains. Cellulases and hemicellulases present in
the enzyme preparation aid in the digestion of the mucilage. A commercial
enzyme preparation is spread on the grains (2–10 g/ton at 15–20ºC). The
fermentation stage of coffee is accelerated, and reduced from 40 or 80, to 20
hr due to enzyme treatment (Carr 1985, Godfrey 1985). Alkaline pectinases
are mainly used to remove fiber and pretreatment of industrial juices, but
they are mostly from bacterial sources. Other application examples would
be the production of Japanese paper, paper manufacture, and oil extraction
(e.g., Canola), as well as, coffee and tea fermentation (Kapoor et al. 2000,
Kashyap et al. 2001, Hoondal et al. 2002).

Extraction of olive oil

In the past few years, olive oil has attracted the world market due to its
numerous benefits for health. Its extraction involves olive crushing in stone
or a knife mill, passage of the resulting paste through a series of horizontal
mixers and decanters and centrifugation at high speed for oil recovery. The
main advantages of using maceration enzymes during olive oil extraction
are: increased extraction (up to 2 Kg oils/100 Kg olives), better fractionation
during centrifugation, obtaining of oil with high levels of antioxidants and
vitamin E and decreased rancidification (Galante and Conti 1998).
Canola seed, coconut, sunflower seed and olive oils are traditionally
produced by the extraction with organic solvent; hexane is the most common
and a potential carcinogenic. Cell wall degrading enzymes including
pectinases are used in the extraction of vegetable oils in aqueous processes
for the liquefaction of the structural components of cell walls of seeds that
contain oil (Kashyap et al. 2001).

Wine production
The first enzyme used in the wine industry was a commercial pectinase
from Aspergillus. Over the last four decades, attempts have been made to
improve yeast strains for fermentation of grape juices as well as microbial
 Pectinases Produced by Microorganisms: Properties and Applications 333

enzymes during wine production. The three major extracellular enzymes

used were pectinases, beta-glucanases and hemicellulases. Their main
benefits included clarification and easier filtration, as well as improved
quality and stability of the wine. Addition of pectinases during grape
crushing improved juice extraction, reduced time for clarification and
increased the terpene content of the wine. Furthermore, pectinases with
high pectin lyases and low pectin methyl esterase activities are preferred
to minimize methanol release from methylpolygalacturonic acid during
production (Galante et al. 1998).

Animal feed
Pectinases are used together with other enzymes to reduce viscosity of
the feed, increase the absorption and release of nutrients via hydrolysis
of non-degradable fibers and of nutrients blocked by fibers (Sharma and
Sathyanarayama 2006).

Textile industry
Pectinolytic enzymes can be used in these industries to degrade the pectin
layer that covers cellulose fibers, releasing them for posterior processing
(Piccoli-Valle et al. 2001), at the treatment of fluid residues and the
degumming of natural fibers (Kaur et al. 2004, Klug-Santner et al. 2006).
Pectinases are used for the maceration of vegetable fibers, linen, hemp and
jute, in the biopreparation of cotton and the enzymatic polishing of mixed
jute and cotton tissue (Kaur et al. 2004, Klug-Santner et al. 2006).
Degumming of rami with pectin lyase produces fibers of higher quality
than those produced by commercial enzyme complexes or using a chemical
process with alkali, and also reduces environmental pollution (Silva et al.
2005). In crude cotton, removal of pectin wax and gumming agents by
the use of pectinases plus amylases, lipases and hemicellulases, under
adequate conditions, replaces the uses of caustic soda, generating high
quality products, for subsequent dyeing and weaving, with lesser energy
consumption (Sawada and Ueda 2001).

Cellulose and paper industries

During paper manufacturing, pectinases may depolymerize pectin-
containing substances and decrease cationic demand of pectic solutions and
result in filtrates from peroxide blanching, solving problems of retention
in mechanical blanching of cellulose and treatment of effluents from paper
mills (Sharma and Sathyanarayama 2006).
334 Fungal Enzymes

Functional foods and pharmaceuticals

The pectin hydrolysates and polysaccharide pectics are classified as
probiotic, since they can be used as promoters of health in human and animal
nutrition, through growth stimulus to select and/or the activity of species
of resident bacteria in the intestinal colon (Uenojo and Pastore 2007).
Yamada (1995) found two different bioactive pectic polysaccharides,
bupleuran 2IIb and 2IIc roots of Bupleurum falcatum. Bupleuran 2IIc,
consisting mainly of galacturonic partial regions, showed potent anti-ulcer
activity. The digestion of bupleuran 2IIc with endo-polygalacturonase
provided mainly galacturonic oligosaccharides and small proportions of
regions resistant to the enzyme. The results of the PG resistant carbohydrate
portion showed a potent activity that is indicative of a role for endo-PG
in the production of pectic poly-pectioligosaccharides as pharmaceuticals
(Lang and Dörnenburg 2000).

Fermentation of tea and coffee

The pectinases have an important role in the fermentation of tea and coffee.
The fermentation of coffee beans using pectinases is used by microorganisms
acting on the removal of the mucilage layer of grains. The stage of the
fermentation process of coffee is fast and reduces from approximately
80 hr to 20 hr with the enzymatic treatment, thereby improving the final
product.
Pectinases from fungi are also used in the manufacturing of tea. The
fermentation of tea treated with enzyme accelerates, although the dose
may carefully be adjusted to avoid damage to the tea leaves (Kashyap
et al. 2001).

Treatment of wastewater pectin

The citrus processing industry uses a certain amount of water that contains
little pectinaceus material decomposed by microorganisms in the treatment
of activated sludge. Tanabe et al. (1987) attempted to develop a new
treatment for wastewater, using processing alkalophillic microorganism that
produces an endo-lyase alkaline extracellular pectinase, and the treatment
with this lineage caused useful results in the removal of pectin substances
from wastewater (Kashyap et al. 2001).

Treatment of the banana fiber

Banana fibers are light weight, soft fibers obtained from the pseudo-stem of
the banana. After harvesting the fruit, the stem of the banana is discarded
 Pectinases Produced by Microorganisms: Properties and Applications 335

as a waste material. The banana fiber can be used for the production of the
Kraft paper (Jacob et al. 2008).
Jocob et al. (2008) performed a study of degradation of the fiber of
dried banana and hand stripped with crude extract of pectinase from the
Streptomyces lydicus lineage and observed that a gradual release of reducing
sugar was an effective treatment.
The excessive removal of the non-cellulolytic coating, gummy material
part of the fiber of the plant cell is called degumming, and is required before
the industrial use of the fiber. The chemical treatment of the fiber is a toxic
waste and causes serious threats to the environment as well as biological
disturbances. Pectinases play the main role for the processing of these
fibers, since 40 percent of dry weight change of the plant cell is comprised
by pectin (Jacob et al. 2008).

Conclusions and Perspectives

From the beginning of polysaccharide research, in the first half of the 20th
century, phenomenal advances in enzyme production and improvement of
their properties for many industrial uses have taken place. Identification of
organisms, mainly with thermophilic characteristics, enables the producing
processes which work at high temperatures and various pH levels, showing
good stability under these conditions. Advances in the field of recombinant
DNA technology provides a means for the super expression of enzymes.
Yeasts are able to provide ease of growth and genetic manipulation, as
well as the capacity to perform specific post-translation modifications for
eukaryotic cells. Furthermore, if compared to more complex eukaryote
systems, yeast cells are more economical, present better yields and require
less time and efforts. Recombinant DNA technology and/or site-directed
mutations might lead to the production of pectinases, and genes might
be easily introduced in selected microorganisms that use this new genetic
material by means of direct expression.
Another great challenge for research is to aggregate value to
agroindustrial residues that are being produced in large scale as sugarcane
refuse, fruit peels or other agro/animal husbandry products, that
when accumulated lead to environmental pollution. The cultivation of
microorganisms, mainly filamentous fungi fermented on solid substrate,
has become a solid basis for pectinase production and other enzymes with
potential industrial application. Thus, the combination of strategies like
the cultivation of thermophile fungi with agro-industrial residues, allied
to the development of enzyme superexpression by molecular techniques,
may benefit various industrial sectors as well as the environment.
336 Fungal Enzymes

Acknowledgements
We thank Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP), Conselho de Desenvolvimento Científico e Tecnológico (CNPq)
and National System for Research on Biodiversity (Sisbiota-Brazil, CNPq
563260/2010-6/FAPESP nº 2010/52322-3). M.L.T.M.P. and H. Cabral are
Research Fellows of CNPq. We also thank Mariana Cereia for English
review.

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